Soybean cultivar CX300

ABSTRACT

The instant invention relates to the novel soybean cultivar designated CX300. Provided by the invention are the seeds, plants and derivatives of the soybean cultivar CX300. Also provided by the invention are tissue cultures of the soybean cultivar CX300 and the plants regenerated therefrom. Still further provided by the invention are methods for producing soybean plants by crossing the soybean cultivar CX300 with itself or another soybean variety, as well as the plants produced by such methods.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of soybeanbreeding. In particular, the invention relates to the novel soybeancultivar CX300.

2. Description of Related Art

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possess the traits to meetthe program goals. The goal is to combine in a single variety animproved combination of desirable traits from the parental germplasm.These important traits may include higher seed yield, resistance todiseases and insects, better stems and roots, tolerance to drought andheat, better agronomic quality, resistance to herbicides, andimprovements in compositional traits.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, recurrent selection andbackcrossing.

The complexity of inheritance influences choice of the breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable cultivar. This approach hasbeen used extensively for breeding disease-resistant cultivars (Bowerset al., 1992; Nickell and Bernard, 1992). Various recurrent selectiontechniques are used to improve quantitatively inherited traitscontrolled by numerous genes. The use of recurrent selection inself-pollinating crops depends on the ease of pollination, the frequencyof successful hybrids from each pollination, and the number of hybridoffspring from each successful cross.

Each breeding program should include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested and compared toappropriate standards in environments representative of the commercialtarget area(s) for generally three or more years. The best lines arecandidates for new commercial cultivars. Those still deficient in a fewtraits may be used as parents to produce new populations for furtherselection.

These processes, which lead to the final step of marketing anddistribution, may take as much as eight to 12 years from the time thefirst cross is made. Therefore, development of new cultivars is atime-consuming process that requires precise forward planning, efficientuse of resources, and a minimum of changes in direction.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to one or more widely grownstandard cultivars. Single observations are generally inconclusive,while replicated observations provide a better estimate of geneticworth.

The goal of plant breeding is to develop new, unique and superiorsoybean cultivars and hybrids. The breeder initially selects and crossestwo or more parental lines, followed by repeated selfing and selection,producing many new genetic combinations. The breeder can theoreticallygenerate billions of different genetic combinations via crossing,selfing and mutations. The breeder has no direct control at the cellularlevel. Therefore, two breeders will never develop the same line de novo,or even very similar lines, having the same soybean traits.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made, during and at the end of the growing season. The cultivarswhich are developed are unpredictable. This unpredictability is becausethe breeder's selection occurs in unique environments, with no controlat the DNA level (using conventional breeding procedures), and withmillions of different possible genetic combinations being generated. Abreeder of ordinary skill in the art cannot predict the final resultinglines he develops, except possibly in a very gross and general fashion.The same breeder cannot produce the same cultivar twice by using theexact same original parents and the same selection techniques. Thisunpredictability results in the expenditure of large amounts of researchmonies to develop superior new soybean cultivars.

The development of new soybean cultivars requires the development andselection of soybean varieties, the crossing of these varieties andselection of progeny from the superior hybrid crosses. The hybrid seedis produced by manual crosses between selected male-fertile parents orby using male sterility systems. Hybrids may be identified by usingcertain single locus traits such as pod color, flower color, pubescencecolor or herbicide resistance which indicate that the seed is truly ahybrid. Additional data on parental lines as well as the phenotype ofthe hybrid influence the breeder's decision whether to continue with thespecific hybrid cross.

Pedigree breeding and recurrent selection breeding methods are used todevelop cultivars from breeding populations. Breeding programs combinedesirable traits from two or more cultivars or various broad-basedsources into breeding pools from which cultivars are developed byselfing and selection of desired phenotypes. The new cultivars areevaluated to determine which have commercial potential.

Pedigree breeding is commonly used for the improvement ofself-pollinating crops. Two parents which possess favorable,complementary traits are crossed to produce an F₁. An F₂ population isproduced by selfing one or several F₁ 's. Selection of the bestindividuals may begin in the F₂ population (or later depending upon thebreeders objectives); then, beginning in the F₃, the best individuals inthe best families can be selected. Replicated testing of families canbegin in the F₃ or F₄ generation to improve the effectiveness ofselection for traits with low heritability. At an advanced stage ofinbreeding (i.e., F₆ and F₇), the best lines or mixtures ofphenotypically similar lines are tested for potential release as newcultivars.

Mass and recurrent selections can be used to improve populations ofeither self-or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Backcross breeding has been used to transfer genetic loci for simplyinherited, highly heritable traits into a desirable homozygous cultivarwhich is the recurrent parent. The source of the trait to be transferredis called the donor or nonrecurent parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the donor parentare selected and repeatedly crossed (backcrossed) to the recurrentparent. The resulting plant is expected to have the attributes of therecurrent parent (e.g., cultivar) and the desirable trait transferredfrom the donor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

In a multiple-seed procedure, soybean breeders commonly harvest one ormore pods from each plant in a population and thresh them together toform a bulk. Part of the bulk is used to plant the next generation andpart is put in reserve. The procedure has been referred to as modifiedsingle-seed descent or the pod-bulk technique.

The multiple-seed procedure has been used to save labor at harvest. Itis considerably faster to thresh pods with a machine than to remove oneseed from each by hand for the single-seed procedure. The multiple-seedprocedure also makes it possible to plant the same number of seeds of apopulation each generation of inbreeding. Enough seeds are harvested tomake up for those plants that did not germinate or produce seed.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Allard, 1960; Simmonds, 1979; Sneep et al., 1979; Fehr,1987a,b).

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, the grower, processor and consumer; for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

Soybean, Glycine max (L), is an important and valuable field crop. Thus,a continuing goal of plant breeders is to develop stable, high yieldingsoybean cultivars that are agronomically sound. The reasons for thisgoal are to maximize the amount of grain produced on the land used andto supply food for both animals and humans. To accomplish this goal, thesoybean breeder must select and develop soybean plants that have thetraits that result in superior cultivars.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to seed of the soybeancultivar designated CX300. The invention also relates to plants producedby growing the seed of the soybean cultivar CX300, as well as thederivatives of such plants. As used herein, the term "plant" includesplant cells, plant protoplasts, plant cells of a tissue culture fromwhich soybean plants can be regenerated, plant calli, plant clumps, andplant cells that are intact in plants or parts of plants, such aspollen, flowers, seeds, pods, leaves, stems, and the like.

Another aspect of the invention relates to a tissue culture ofregenerable cells of the soybean cultivar CX300, as well as plantsregenerated therefrom, wherein the regenerated soybean plant is capableof expressing all the physiological and morphological characteristics ofa plant grown from the soybean seed designated CX300.

Yet another aspect of the current invention is a soybean plantcomprising a single locus conversion of the soybean cultivar CX300,wherein the soybean plant is otherwise capable of expressing all thephysiological and morphological characteristics of the soybean cultivarCX300. In particular embodiments of the invention, the single locusconversion may comprise a transgenic gene which has been introduced bygenetic transformation into the soybean cultivar CX300 or a progenitorthereof. In still other embodiments of the invention, the single locusconversion may comprise a dominant or recessive allele. The locusconversion may confer potentially any trait upon the single locusconverted plant, including herbicide resistance, insect resistance,resistance to bacterial, fungal, or viral disease, male fertility orsterility, and improved nutritional quality.

Still yet another aspect of the invention relates to a first generation(F₁) hybrid soybean seed produced by crossing a plant of the soybeancultivar CX300 to a second soybean plant. Also included in the inventionare the F₁ hybrid soybean plants grown from the hybrid seed produced bycrossing the soybean cultivar CX300 to a second soybean plant. Stillfurther included in the invention are the seeds of an F₁ hybrid plantproduced with the soybean cultivar CX300 as one parent, the secondgeneration (F₂) hybrid soybean plant grown from the seed of the F₁hybrid plant, and the seeds of the F₂ hybrid plant.

Still yet another aspect of the invention is a method of producingsoybean seeds comprising crossing a plant of the soybean cultivar CX300to any second soybean plant, including itself or another plant of thecultivar CX300. In particular embodiments of the invention, the methodof crossing comprises the steps of a) planting seeds of the soybeancultivar CX300; b) cultivating soybean plants resulting from said seedsuntil said plants bear flowers; c) allowing fertilization of the flowersof said plants; and, d) harvesting seeds produced from said plants.

Still yet another aspect of the invention is a method of producinghybrid soybean seeds comprising crossing the soybean cultivar CX300 to asecond, distinct soybean plant which is nonisogenic to the soybeancultivar CX300. In particular embodiments of the invention, the crossingcomprises the steps of a) planting seeds of soybean cultivar CX300 and asecond, distinct soybean plant, b) cultivating the soybean plants grownfrom the seeds until the plants bear flowers; c) cross pollinating aflower on one of the two plants with the pollen of the other plant, andd) harvesting the seeds resulting from the cross pollinating.

DETAILED DESCRIPTION OF THE INVENTION

The instant invention provides methods and composition relating toplants, seeds and derivatives of the soybean cultivar CX300. Soybeancultivar CX300 has superior characteristics and is derived from thecross CX250b×CX267. The origin and breeding history of CX300 were asfollows:

Summer 1992 The cross CX250b×CX267 was made.

Winter 1992-93 F1 generation was grown (range 1, row 5). F2 generationwas grown (range 5, rows 1-10).

Summer 1993 F3 generation was grown (range 614, rows 21-40 through range615, rows 1-20).

Winter 1993-94 F4 generation was grown. Range 34, row 71 was selected.

Summer 1994 F5 generation was grown.

Summer 1995 F6 generation was grown.

Winter 1995-96 F7 generation was grown.

Summer 1996 F8 generation was grown.

Summer 1997 F9 generation was grown.

Summer 1998 F10 generation was grown.

Winter 1998-99 F11 seed was released as CX300.

The results of an objective description of the variety, based on datacollected at Bloomington, Ill., were as follows:

    ______________________________________                                        SEED SHAPE:           Spherical                                                 SEED COAT COLOR (Mature Seed): Yellow                                         SEED COAT LUSTER(Mature Hand Shelled Dull                                     Seed):                                                                        SEED SIZE (Mature Seed): 15.3 g/100 seed                                      HILUM COLOR: (Mature Seed) Buff                                               COTYLEDON COLOR (Mature Seed): Yellow                                         SEED PROTEIN PEROXIDASE ACTIVITY: High                                        SEED PROTEIN ELECTROPHORECTIC --                                              BAND:                                                                         HYPOCOTYL COLOR: Green with Bronze Band                                       LEAFLET SHAPE: Ovate                                                          LEAFLET SIZE: Medium                                                          LEAF COLOR: Medium Green                                                      FLOWER COLOR: White                                                           POD COLOR: Tan                                                                PLANT PUBESCENCE COLOR: Gray                                                  PLANT TYPE: Medium                                                            PLANT HABIT: Indeterminate                                                    MATURITY GROUP: III                                                         ______________________________________                                    

    ______________________________________                                        DISEASE REACTION: (0 = NOT TESTED; 1 = SUSCEPTIBLE;                             2 = RESISTANT)                                                              ______________________________________                                          BACTERIAL DISEASES    FUNGAL DISEASES                                         Bacterial Pustule: 0 Brown Spot: 0                                            Bacterial Blight: 0 Frogeye Leaf Spot: 0                                      Wildfire: 0 Target Spot: 0                                                      Downy Mildew: 0                                                             VIRAL DISEASES  Powdery Mildew: 0                                             Bud Blight: 0 Brown Stem Rot: 2*                                              Yellow Mosaic: 0 Stem Canker: 0                                               Cowpea Mosaic: 0 Pod and Stem Blight: 0                                       Pod Mottle: 0 Purple Seed Stain: 0                                            Seed Mottle: 0 Rhizoctonia Root Rot: 0                                          Sclerotinia White Intermediate**                                              Mold:                                                                         Sudden Death 2***                                                             Syndrome:                                                                   NEMATODE DISEASES  Phytophthora Rot: 2 (Rpslc)                              Soybean Cyst Nematode:                                                                      1     Race(s): Race 1:                                                                              0                                           Lance Nematode: 0  Race 2: 0                                                  Southern Root Knot 0  Race 3: 0                                               Nematode:                                                                     Northern Root Knot 0  Race 4: 0                                               Nematode:                                                                     Peanut Root Knot 0  Race 5-9: 0                                               Nematode:                                                                     Reniform Nematode: 0                                                        ______________________________________                                        *DEKALB's score for Brown Stem Rot is 2 (Rating scale 1-9:                      1 = most resistant)                                                           **DEKALB's score for Sclerotinia White Mold is 5 (Rating scale 1-9:          1 = most resistant)                                                           ***DEKALB's score for Sudden Death Syndrome is 2 (Rating scale 1-9:           1 = most resistant)                                                          ______________________________________                                          PHYSIOLOGICAL RESPONSES: (0 = NOT TESTED;                                      1 = SUSCEPTIBLE; 2 = RESISTANT)                                            ______________________________________                                              Iron Chlorosis on Calcareous                                                                   Intermediate*                                            Soil:                                                                         Other: 0                                                                    ______________________________________                                        *DEKALB's score for Iron Chlorosis is 5 (Rating scale 1-9:                      1 = most resistant)                                                         ______________________________________                                          INSECT REACTION: (0 = NOT TESTED; 1 = SUSCEPTIBLE;                            2 = RESISTANT)                                                              ______________________________________                                              Mexican Bean Beetle:                                                                           0                                                        Potato Leaf Hopper: 0                                                         Other: 0                                                                    ______________________________________                                    

Soybean variety CX300 has been judged to be uniform for breedingpurposes and testing after four generations of selfing. CX300 wasreproduced and judged uniform and stable for an additional sevengenerations. Cultivar CX300 shows no variants other than what wouldnormally be expected due to environment or that would occur for almostany characteristic during the course of repeated sexual reproduction.Some of the criteria used to select in various generations include: seedyield, lodging resistance, emergence, seedling vigor, disease tolerance,maturity, plant height and shattering resistance.

The inventor believes that CX300 is most similar to soybean varietyCX278, however, the varieties differ for at least the following traits:CX300 has white flowers, buff hila and is resistant to brown stem rotwhereas, CX278 has purple flowers, gray hila and is susceptible to brownstem rot.

I. Variety Comparison

Direct comparisons were made between CX300 and the competing commercialvariety CX278. Traits measured included yield, maturity, moisture,lodging, plant height, field emergence, and seedling vigor. The resultsof the comparison are presented in below. Shown are the number of testsin which the varieties were compared, the deviation or difference of theresults, the test means, and the traits which showed a significantdifference and the significance level.

    __________________________________________________________________________    VARIETY COMPARISON                                                              CX300                                                                       VARIETIES SEL                                                                              YLD MAT                                                                              MST LDG                                                                              PLTHT                                                                              FEMR                                                                              SVIG                                        COMPARED TYP % M B/A DAY % RAT IN RAT RAT                                   __________________________________________________________________________    CX300  R  100.3                                                                            52.8                                                                              268.9                                                                            10.3                                                                              2.6                                                                              33.4 2.7 3.0                                          50                                                                           CX278  100.3 51.0 267.5 10.5 3.3 36.3 2.6 3.0                                 Deviation  0.0  1.8  1.5 -0.2 -0.7  -2.9 0.1 0.0                              TestMean  100.0 50.1 268.0 10.5 4.0 34.0 3.6 3.7                              Sig   * ** ** ** *                                                          __________________________________________________________________________     Signficance levels are indicated as: + = 10%, * = 5%, ** = 1%                 TYP = Research  No. of tests                                                  SEL % M = Selection Index (% test mean)                                       YLD B/A = Yield (bushels /acre)                                               MAT DAY = Maturity (days)                                                     MST % = Moisture (percentage)                                                 LDG RAT = Lodging Rating (scale: 1-9, 1 = best)                               PLTHT IN = Plant Height (inches)                                              FEMR RAT = Field Emergence Rating (scale: 1-9, 1 = best)                      SVIG RAT = Seedling Vigor Rating (scale: 1-9, 1 = best)                  

II. Breeding Soybean Cultivar CX300

An important aspect of the invention concerns methods for crossing thesoybean cultivar CX300 with itself or a second plant and the seeds andplants produced by such methods. These methods can be used forpropagation of the soybean cultivar CX300, or can be used to producehybrid soybean seeds and the plants grown therefrom. Hybrid soybeanplants can be used by farmers in the commercial production of soyproducts or may be advanced in certain breeding protocols for theproduction of novel soybean cultivars. The cultivar of the presentinvention is well suited to the development of new cultivars because ofthe elite nature of it's genetic background. A hybrid plant can also beused as a recurrent parent at any given stage in a backcrossing protocolduring the production of a single locus conversion of the soybeancultivar CX300.

In selecting a second plant to cross with CX300 for the purpose ofdeveloping novel soybean cultivars, it will typically be desired tochoose those plants which either themselves exhibit one or more selecteddesirable characteristics or which exhibit the desired characteristic(s)when in hybrid combination. Examples of potentially desiredcharacteristics include seed yield, lodging resistance, emergence,seedling vigor, disease tolerance, maturity, plant height and shatteringresistance.

Any time the soybean cultivar CX300 is crossed with another, different,cultivar, first generation (F₁) soybean progeny are produced. The hybridprogeny are produced regardless of characteristics of the two cultivarsproduced. As such, an F₁ hybrid soybean plant may be produced bycrossing CX300 with any second soybean plant. The second soybean plantmay be genetically homogeneous (e.g., inbred) or may itself be a hybrid.Therefore, any F₁ hybrid soybean plant produced by crossing soybeancultivar CX300 with a second soybean plant is a part of the presentinvention.

Soybean plants (Glycine max L.) can be crossed by either natural ormechanical techniques (see, e.g., Fehr, 1980). Natural pollinationoccurs in soybeans either by self pollination or natural crosspollination, which typically is aided by pollinating organisms. Ineither natural or artificial crosses, flowering and flowering time arean important consideration. Soybean is a short-day plant, but there isconsiderable genetic variation for sensitivity to photoperiod (Hamner,1969; Criswell and Hume, 1972). The critical day length for floweringranges from about 13 h for genotypes adapted to tropical latitudes to 24h for photoperiod-insensitive genotypes grown at higher latitudes(Shibles et al., 1975). Soybeans seem to be insensitive to day lengthfor 9 days after emergence. Photoperiods shorter than the critical daylength are required for 7 to 26 days to complete flower induction(Borthwick and Parker, 1938; Shanmugasundaram and Tsou, 1978).

Sensitivity to day length is an important consideration when genotypesare grown outside of their area of adaptation. When genotypes adapted totropical latitudes are grown in the field at higher latitudes, they maynot mature before frost occurs. Plants can be induced to flower andmature earlier by creating artificially short days or by grafting (Fehr,1980). Soybeans frequently are grown in winter nurseries located at sealevel in tropical latitudes where day lengths are much shorter thantheir critical photoperiod. The short day lengths and warm temperaturesencourage early flowering and seed maturation, and genotypes can producea seed crop in 90 days or fewer after planting. Early flowering isuseful for generation advance when only a few self-pollinated seeds perplant are needed, but not for artificial hybridization because theflowers self-pollinate before they are large enough to manipulate forhybridization. Artificial lighting can be used to extend the natural daylength to about 14.5 h to obtain flowers suitable for hybridization andto increase yields of self-pollinated seed.

The effect of a short photoperiod on flowering and seed yield can bepartly offset by altitude, probably due to the effects of cooltemperature (Major et al., 1975). At tropical latitudes, cultivarsadapted to the northern U.S. perform more like those adapted to thesouthern U.S. at high altitudes than they do at sea level.

The light level required to delay flowering is dependent on the qualityof light emitted from the source and the genotype being grown. Bluelight with a wavelength of about 480 nm requires more than 30 times theenergy to inhibit flowering as red light with a wavelength of about 640nm (Parker et al., 1946).

Temperature can also play a significant role in the flowering anddevelopment of soybean (Major et al., 1975). It can influence the timeof flowering and suitability of flowers for hybridization. Temperaturesbelow 21° C. or above 32° C. can reduce floral initiation or seed set(Hamner, 1969; van Schaik and Probst, 1958). Artificial hybridization ismost successful between 26° C. and 32° C. because cooler temperaturesreduce pollen shed and result in flowers that self-pollinate before theyare large enough to manipulate. Warmer temperatures frequently areassociated with increased flower abortion caused by moisture stress;however, successful crosses are possible at about 35° C. if soilmoisture is adequate.

Soybeans have been classified as indeterminate, semi-determinate, anddeterminate based on the abruptness of stem termination after floweringbegins (Bernard and Weiss, 1973). When grown at their latitude ofadaptation, indeterminate genotypes flower when about one-half of thenodes on the main stem have developed. They have short racemes with fewflowers, and their terminal node has only a few flowers.Semi-determinate genotypes also flower when about one-half of the nodeson the main stem have developed, but node development and flowering onthe main stem stops more abruptly than on indeterminates. Their racemesare short and have few flowers, except for the terminal one, which mayhave several times more flowers than those lower on the plant.Determinate cultivars begin flowering when all or most of the nodes onthe main stem have developed. They usually have elongated racemes thatmay be several centimeters in length and may have a large number offlowers. Stem termination and flowering habit are reported to becontrolled by two major genes (Bernard and Weiss, 1973).

Soybean flowers typically are self-pollinated on the day the corollaopens. The amount of natural crossing, which is typically associatedwith insect vectors such as honeybees, is approximately 1% for adjacentplants within a row and 0.5% between plants in adjacent rows. Thestructure of soybean flowers is similar to that of other legume speciesand consists of a calyx with five sepals, a corolla with five petals, 10stamens, and a pistil (Carlson, 1973). The calyx encloses the corollauntil the day before anthesis. The corolla emerges and unfolds to exposea standard, two wing petals, and two keel petals. An open flower isabout 7 mm long from the base of the calyx to the tip of the standardand 6 mm wide across the standard. The pistil consists of a single ovarythat contains one to five ovules, a style that curves toward thestandard, and a club-shaped stigma. The stigma is receptive to pollenabout 1 day before anthesis and remains receptive for 2 days afteranthesis, if the flower petals are not removed. Filaments of ninestamens are fused, and the one nearest the standard is free. The stamensform a ring below the stigma until about 1 day before anthesis, thentheir filaments begin to elongate rapidly and elevate the anthers aroundthe stigma. The anthers dehisce on the day of anthesis, pollen grainsfall on the stigma, and within 10 h the pollen tubes reach the ovary andfertilization is completed (Johnson and Bernard, 1963).

Self-pollination occurs naturally in soybean with no manipulation of theflowers. For the crossing of two soybean plants, it is typicallypreferable, although not required, to utilize artificial hybridization.In artificial hybridization, the flower used as a female in a cross ismanually cross pollinated prior to maturation of pollen from the flower,thereby preventing self fertilization, or alternatively, the male partsof the flower are emasculated using a technique known in the art.Techniques for emasculating the male parts of a soybean flower include,for example, physical removal of the male parts, use of a genetic factorconferring male sterility, and application of a chemical gametocide tothe male parts.

For artificial hybridization employing emasculation, flowers that areexpected to open the following day are selected on the female parent.The buds are swollen and the corolla is just visible through the calyxor has begun to emerge. Usually no more than two buds on a parent plantare prepared, and all self-pollinated flowers or immature buds areremoved with forceps. Special care is required to remove immature budsthat are hidden under the stipules at the leaf axil, and could developinto flowers at a later date. The flower is grasped between the thumband index finger and the location of the stigma determined by examiningthe sepals. A long, curvy sepal covers the keel, and the stigma is onthe opposite side of the flower. The calyx is removed by grasping asepal with the forceps, pulling it down and around the flower, andrepeating the procedure until the five sepals are removed. The exposedcorolla is removed by grasping it just above the calyx scar, thenlifting and wiggling the forceps simultaneously. Care is taken to graspthe corolla low enough to remove the keel petals without injuring thestigma. The ring of anthers is visible after the corolla is removed,unless the anthers were removed with the petals. Cross-pollination canthen be carried out using, for example, petri dishes or envelopes inwhich male flowers have been collected. Desiccators containing calciumchloride crystals are used in some environments to dry male flowers toobtain adequate pollen shed.

It has been demonstrated that emasculation is unnecessary to preventself-pollination (Walker et al., 1979). When emasculation is not used,the anthers near the stigma frequently are removed to make it clearlyvisible for pollination. The female flower usually is hand-pollinatedimmediately after it is prepared; although a delay of several hours doesnot seem to reduce seed set. Pollen shed typically begins in the morningand may end when temperatures are above 30° C., or may begin later andcontinue throughout much of the day with more moderate temperatures.

Pollen is available from a flower with a recently opened corolla, butthe degree of corolla opening associated with pollen shed may varyduring the day. In many environments, it is possible to collect maleflowers and use them immediately without storage. In the southern U.S.and other humid climates, pollen shed occurs in the morning when femaleflowers are more immature and difficult to manipulate than in theafternoon, and the flowers may be damp from heavy dew. In thosecircumstances, male flowers are collected into envelopes or petri dishesin the morning and the open container is typically placed in adesiccator for about 4 h at a temperature of about 25° C. The desiccatormay be taken to the field in the afternoon and kept in the shade toprevent excessive temperatures from developing within it. Pollenviability can be maintained in flowers for up to 2 days when stored atabout 5° C. In a desiccator at 3° C., flowers can be stored successfullyfor several weeks; however, cultivars may differ in the percentage ofpollen that germinates after long-term storage (Kuehl, 1961).

Either with or without emasculation of the female flower, handpollination can be carried out by removing the stamens and pistil with aforceps from a flower of the male parent and gently brushing the anthersagainst the stigma of the female flower. Access to the stamens can beachieved by removing the front sepal and keel petals, or piercing thekeel with closed forceps and allowing them to open to push the petalsaway. Brushing the anthers on the stigma causes them to rupture, and thehighest percentage of successful crosses is obtained when pollen isclearly visible on the stigma. Pollen shed can be checked by tapping theanthers before brushing the stigma. Several male flowers may have to beused to obtain suitable pollen shed when conditions are unfavorable, orthe same male may be used to pollinate several flowers with good pollenshed.

When male flowers do not have to be collected and dried in a desiccator,it may be desired to plant the parents of a cross adjacent to eachother. Plants usually are grown in rows 65 to 100 cm apart to facilitatemovement of personnel within the field nursery. Yield of self-pollinatedseed from an individual plant may range from a few seeds to more than1,000 as a function of plant density. A density of 30 plants/m of rowcan be used when 30 or fewer seeds per plant is adequate, 10 plants/mcan be used to obtain about 100 seeds/plant, and 3 plants/m usuallyresults in maximum seed production per plant. Densities of 12 plants/mor less commonly are used for artificial hybridization.

Multiple planting dates about 7 to 14 days apart usually are used tomatch parents of different flowering dates. When differences inflowering dates are extreme between parents, flowering of the laterparent can be hastened by creating an artificially short day orflowering of the earlier parent can be delayed by use of artificiallylong days or delayed planting. For example, crosses with genotypesadapted to the southern U.S. are made in northern U.S. locations bycovering the late genotype with a box, large can, or similar containerto create an artificially short photoperiod of about 12 h for about 15days beginning when there are three nodes with trifoliate leaves on themain stem. Plants induced to flower early tend to have flowers thatself-pollinate when they are small and can be difficult to prepare forhybridization.

Grafting can be used to hasten the flowering of late floweringgenotypes. A scion from a late genotype grafted on a stock that hasbegun to flower will begin to bloom up to 42 days earlier than normal(Kiihl et al., 1977). First flowers on the scion appear from 21 to 50days after the graft.

Genetic male sterility is available in soybeans and may be useful tofacilitate hybridization in the context of the current invention,particularly for recurrent selection programs (Brim and Stuber, 1973).The distance required for complete isolation of a crossing block is notclear; however, outcrossing is less than 0.5% when male-sterile plantsare 12 m or more from a foreign pollen source (Boerma and Moradshahi,1975). Plants on the boundaries of a crossing block probably sustain themost outcrossing with foreign pollen and can be eliminated at harvest tominimize contamination.

Cross-pollination is more common within rows than between adjacent rows;therefore, it may be preferable to grow populations with genetic malesterility on a square grid to create rows in all directions. Forexample, single-plant hills on 50-cm centers may be used, withsubdivision of the area into blocks of an equal number of hills forharvest from bulks of an equal amount of seed from male-sterile plantsin each block to enhance random pollination.

Observing pod development 7 days after pollination generally is adequateto identify a successful cross. Abortion of pods and seeds can occurseveral weeks after pollination, but the percentage of abortion usuallyis low if plant stress is minimized (Shibles et al., 1975). Pods thatdevelop from artificial hybridization can be distinguished fromself-pollinated pods by the presence of the calyx scar, caused byremoval of the sepals. The sepals begin to fall off as the pods mature;therefore, harvest should be completed at or immediately before the timethe pods reach their mature color. Harvesting pods early also avoids anyloss by shattering.

Once harvested, pods are typically air-dried at not more than 38° C.until the seeds contain 13% moisture or less, then the seeds are removedby hand. Seed can be stored satisfactorily at about 25° C. for up to ayear if relative humidity is 50% or less. In humid climates, germinationpercentage declines rapidly unless the seed is dried to 7% moisture andstored in an air-tight container at room temperature. Long-term storagein any climate is best accomplished by drying seed to 7% moisture andstoring it at 10° C. or less in a room maintained at 50% relativehumidity or in an air-tight container.

III. Single Locus Conversions

When the term soybean cultivar CX300 is used in the context of thepresent invention, this also includes any single locus conversions ofthat cultivar. The term single locus converted plant as used hereinrefers to those soybean plants which are developed by a plant breedingtechnique called backcrossing, wherein essentially all of the desiredmorphological and physiological characteristics of a cultivar arerecovered in addition to the single locus transferred into the cultivarvia the backcrossing technique. Backcrossing methods can be used withthe present invention to improve or introduce a characteristic into thepresent cultivar. The term backcrossing as used herein refers to therepeated crossing of a hybrid progeny back to one of the parentalsoybean plants for that hybrid. The parental soybean plant whichcontributes the locus for the desired characteristic is termed thenonrecurrent or donor parent. This terminology refers to the fact thatthe nonrecurrent parent is used one time in the backcross protocol andtherefore does not recur. The parental soybean plant to which the locusor loci from the nonrecurrent parent are transferred is known as therecurrent parent as it is used for several rounds in the backcrossingprotocol (Poehlman et al., 1995; Fehr, 1987a,b; Sprague and Dudley,1988).

In a typical backcross protocol, the original cultivar of interest(recurrent parent) is crossed to a second cultivar (nonrecurrent parent)that carries the single locus of interest to be transferred. Theresulting progeny from this cross are then crossed again to therecurrent parent and the process is repeated until a soybean plant isobtained wherein essentially all of the desired morphological andphysiological characteristics of the recurrent parent are recovered inthe converted plant, in addition to the single transferred locus fromthe nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalcultivar. To accomplish this, a single locus of the recurrent cultivaris modified or substituted with the desired locus from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphologicalconstitution of the original cultivar. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross; one ofthe major purposes is to add some commercially desirable, agronomicallyimportant trait to the plant. The exact backcrossing protocol willdepend on the characteristic or trait being altered to determine anappropriate testing protocol. Although backcrossing methods aresimplified when the characteristic being transferred is a dominantallele, a recessive allele may also be transferred. In this instance itmay be necessary to introduce a test of the progeny to determine if thedesired characteristic has been successfully transferred.

Soybean cultivars can also be developed from more than two parents(Fehr, 1987a). The technique, known as modified backcrossing, usesdifferent recurrent parents during the backcrossing. Modifiedbackcrossing may be used to replace the original recurrent parent with acultivar having certain more desirable characteristics or multipleparents may be used to obtain different desirable characteristics fromeach.

Many single locus traits have been identified that are not regularlyselected for in the development of a new inbred but that can be improvedby backcrossing techniques. Single locus traits may or may not betransgenic; examples of these traits include, but are not limited to,male sterility, herbicide resistance, resistance to bacterial, fungal,or viral disease, insect resistance, restoration of male fertility,enhanced nutritional quality, yield stability, and yield enhancement.These comprise genes generally inherited through the nucleus.

Direct selection may be applied where the single locus acts as adominant trait. An example of a dominant trait is the herbicideresistance trait. For this selection process, the progeny of the initialcross are sprayed with the herbicide prior to the backcrossing. Thespraying eliminates any plants which do not have the desired herbicideresistance characteristic, and only those plants which have theherbicide resistance gene are used in the subsequent backcross. Thisprocess is then repeated for all additional backcross generations.

One type of single locus trait having particular utility is a gene whichconfers resistance to the herbicide glyphosate. Glyphosate inhibits theaction of the enzyme EPSPS, which is active in the biosynthetic pathwayof aromatic amino acids. Inhibition of this enzyme leads to starvationfor the amino acids phenylalanine, tyrosine, and tryptophan andsecondary metabolites derived therefrom. Mutants of this enzyme areavailable which are resistant to glyphosate. For example, U.S. Pat. No.4,535,060 describes the isolation of EPSPS mutations which conferglyphosate resistance upon organisms having the Salmonella typhimuriumgene for EPSPS, termed aroA. A mutant EPSPS gene having similarmutations also has been cloned from Zea mays. The mutant gene encodes aprotein with amino acid changes at residues 102 and 106. When these orother similar genes are introduced into a plant by genetictransformation, a herbicide resistant phenotype results.

Plants having inherited a transgene comprising a mutated EPSPS gene,may, therefore, be directly treated with the herbicide glyphosatewithout the result of significant damage to the plant. This phenotypeprovides farmers with the benefit of controlling weed growth in a fieldof plants having the herbicide resistance trait by application of thebroad spectrum herbicide glyphosate. For example, one could apply theherbicide ROUNDUP™, a commercial formulation of glyphosate manufacturedand sold by the Monsanto Company, over the top in fields where theglyphosate resistant soybeans are grown. The herbicide application ratesmay range from about 4 ounces of ROUNDUP™ to about 256 ounces ROUNDUP™per acre. More preferably, about 16 ounces to about 64 ounces per acreof ROUNDUP™ may be applied to the field. However, the application ratemay be increased or decreased as needed, based on the abundance and/ortype of weeds being treated. Additionally, depending on the location ofthe field and weather conditions, which will influence weed growth andthe type of weed infestation, it may be desirable to conduct furtherglyphosate treatments. The second glyphosate application will alsotypically comprise an application of about 16 ounces to about 64 ouncesof ROUNDUP™ per acre treated. Again, the treatment rate may be adjustedbased on field conditions. Such methods of application of herbicides toagricultural crops are well known in the art and are summarized ingeneral in Anderson, 1983.

It will be understood to those of skill in the art that a herbicideresistance gene locus may be used for direct selection of plants havingthe resistance gene. For example, by applying about 16 to 64 ounces ofROUNDUP™ per acre to a collection of soybean plants which either have orlack the herbicide resistance trait, the plants lacking the trait willbe killed or damaged. In this way, the herbicide resistant plants may beselected and used for commercial applications or advanced in certainbreeding protocols. This application may find particular use during thebreeding and development of herbicide resistant elite soybean cultivars.

White flower color is an example of a recessive single locus trait. Inthis example, the progeny resulting from the first backcross generation(BC₁) are grown and selfed. The selfed progeny from the BC₁ plant aregrown to determine which BC₁ plants carry the recessive gene for whiteflower color. In other recessive traits, additional progeny testing, forexample growing additional generations such as the BC₁ F₂, may berequired to determine which plants carry the recessive gene.

Selection of soybean plants for breeding is not necessarily dependent onthe phenotype of a plant and instead can be based on geneticinvestigations. For example, one may utilize a suitable genetic markerwhich is closely genetically linked to a trait of interest. One of thesemarkers may therefore be used to identify the presence or absence of atrait in the offspring of a particular cross, and hence may be used inselection of progeny for continued breeding. This technique may commonlybe referred to as marker assisted selection. Any other type of geneticmarker or other assay which is able to identify the relative presence orabsence of a trait of interest in a plant may also be useful forbreeding purposes. Exemplary procedures for marker assisted selectionwhich are applicable to the breeding of soybeans are disclosed U.S. Pat.No. 5,437,697, and U.S. Pat. No. 5,491,081, both of which disclosuresare specifically incorporated herein by reference in their entirety.Such methods will be of particular utility in the case of recessivetraits and variable phenotypes, or where conventional assays areexpensive, time consuming or otherwise disadvantageous. Types of geneticmarkers which could be used in accordance with the invention include,for example, Simple Sequence Length Polymorphisms (SSLPs) (Williams etal., 1990), Randomly Amplified Polymorphic DNAs (RAPDs), DNAAmplification Fingerprinting (DAF), Sequence Characterized AmplifiedRegions (SCARs), Arbitrary Primed Polymerase Chain Reaction (AP-PCR),Amplified Fragment Length Polymorphisms (AFLPs) (EP 534 858,specifically incorporated herein by reference in its entirety), andSingle Nucleotide Polymorphisms (SNPs) (Wang et al., 1998).

Many qualitative characters also have potential use as phenotype-basedgenetic markers in soybeans; however, some or many may not differ amongcultivars commonly used as parents (Bernard and Weiss, 1973). The mostwidely used genetic markers are flower color (purple dominant to white),pubescence color (brown dominant to gray), and pod color (brown dominantto tan). The association of purple hypocotyl color with purple flowersand green hypocotyl color with white flowers is commonly used toidentify hybrids in the seedling stage. Differences in maturity, height,hilum color, and pest resistance between parents can also be used toverify hybrid plants.

IV. Origin and Breeding History of an Exemplary Single Locus ConvertedPlant

The soybean cultivar known as Williams '82 [Glycine max L. Merr.] (Reg.No. 222, PI 518671) was developed using backcrossing techniques totransfer a locus comprising the Rps₁. gene to the cultivar Williams(Bernard and Cremeens, 1988). Williams '82 is a composite of fourresistant lines from the BC₆ F₃ generation, which were selected from 12field-tested resistant lines from Williams X Kingwa. The cultivarWilliams was used as the recurrent parent in the backcross and thecultivar Kingwa was used as the source of the Rps₁ locus. This genelocus confers resistance to 19 of the 24 races of the fungal agentphytopthora rot.

The F₁ or F₂ seedlings from each backcross round were tested forresistance to the fungus by hypocotyl inoculation using the inoculum ofrace 5. The final generation was tested using inoculum of races 1 to 9.In a backcross such as this, where the desired characteristic beingtransferred to the recurrent parent is controlled by a major gene whichcan be readily evaluated during the backcrossing, it is common toconduct enough backcrosses to avoid testing individual progeny forspecific traits such as yield in extensive replicated tests. In general,four or more backcrosses are used when there is no evaluation of theprogeny for specific traits, such as yield. As in this example, lineswith the phenotype of the recurrent parent may be composited without theusual replicated tests for traits such as yield, protein or oilpercentage in the individual lines.

The cultivar Williams '82 is comparable to the recurrent parent cultivarWilliams in all traits except resistance to phytopthora rot. Forexample, both cultivars have a relative maturity of 38, indeterminatestems, white flowers, brown pubescence, tan pods at maturity and shinyyellow seeds with black to light black hila.

V. Tissue Cultures and in vitro Regeneration of Soybean Plants

A further aspect of the invention relates to tissue cultures of thesoybean cultivar designated CX300. As used herein, the term "tissueculture" indicates a composition comprising isolated cells of the sameor a different type or a collection of such cells organized into partsof a plant. Exemplary types of tissue cultures are protoplasts, calliand plant cells that are intact in plants or parts of plants, such asembryos, pollen, flowers, leaves, roots, root tips, anthers, and thelike. In a preferred embodiment, the tissue culture comprises embryos,protoplasts, meristematic cells, pollen, leaves or anthers.

Exemplary procedures for preparing tissue cultures of regenerablesoybean cells, for example, from soybean cultivar CX300, andregenerating soybean plants therefrom, are disclosed in U.S. Pat. No.4,992,375; U.S. Pat. No. 5,015,580; U.S. Pat. No. 5,024,944, and U.S.Pat. No. 5,416,011, each of the disclosures of which is specificallyincorporated herein by reference in its entirety.

An important ability of a tissue culture is the capability to regeneratefertile plants. This allows, for example, transformation of the tissueculture cells followed by regeneration of transgenic plants. Fortransformation to be efficient and successful, DNA must be introducedinto cells that give rise to plants or germ-line tissue.

Soybeans typically are regenerated via two distinct processes; shootmorphogenesis and somatic embryogenesis (Finer, 1996). Shootmorphogenesis is the process of shoot meristem organization anddevelopment. Shoots grow out from a source tissue and are excised androoted to obtain an intact plant. During somatic embryogenesis, anembryo (similar to the zygotic embryo), containing both shoot and rootaxes, is formed from somatic plant tissue. An intact plant rather than arooted shoot results from the germination of the somatic embryo.

Shoot morphogenesis and somatic embryogenesis are different processesand the specific route of regeneration is primarily dependent on theexplant source and media used for tissue culture manipulations. Whilethe systems are different, both systems show cultivar-specific responseswhere some lines are more responsive to tissue culture manipulationsthan others. A line that is highly responsive in shoot morphogenesis maynot generate many somatic embryos. Lines that produce large numbers ofembryos during an `induction` step may not give rise to rapidly-growingproliferative cultures. Therefore, it may be desired to optimize tissueculture conditions for each soybean line. These optimizations mayreadily be carried out by one of skill in the art of tissue culturethrough small-scale culture studies. In addition to line-specificresponses, proliferative cultures can be observed with both shootmorphogenesis and somatic embryogenesis. Proliferation is beneficial forboth systems, as it allows a single, transformed cell to multiply to thepoint that it will contribute to germ-line tissue.

Shoot morphogenesis was first reported by Wright et al. (1986) as asystem whereby shoots were obtained de novo from cotyledonary nodes ofsoybean seedlings. The shoot meristems were formed subepidermally andmorphogenic tissue could proliferate on a medium containing benzyladenine (BA). This system can be used for transformation if thesubepidermal, multicellular origin of the shoots is recognized andproliferative cultures are utilized. The idea is to target tissue thatwill give rise to new shoots and proliferate those cells within themeristematic tissue to lessen problems associated with chimerism.Formation of chimeras, resulting from transformation of only a singlecell in a meristem, are problematic if the transformed cell is notadequately proliferated and does not does not give rise to germ-linetissue. Once the system is well understood and reproducedsatisfactorily, it can be used as one target tissue for soybeantransformation.

Somatic embryogenesis in soybean was first reported by Christianson etal. (1983) as a system in which embryogenic tissue was initiallyobtained from the zygotic embryo axis. These embryogenic cultures wereproliferative but the repeatability of the system was low and the originof the embryos was not reported. Later histological studies of adifferent proliferative embryogenic soybean culture showed thatproliferative embryos were of apical or surface origin with a smallnumber of cells contributing to embryo formation. The origin of primaryembryos (the first embryos derived from the initial explant) isdependent on the explant tissue and the auxin levels in the inductionmedium (Hartweck et al., 1988). With proliferative embryonic cultures,single cells or small groups of surface cells of the `older` somaticembryos form the `newer` embryos.

Embryogenic cultures can also be used successfully for regeneration,including regeneration of transgenic plants, if the origin of theembryos is recognized and the biological limitations of proliferativeembryogenic cultures are understood. Biological limitations include thedifficulty in developing proliferative embryogenic cultures and reducedfertility problems (culture-induced variation) associated with plantsregenerated from long-term proliferative embryogenic cultures. Some ofthese problems are accentuated in prolonged cultures. The use of morerecently cultured cells may decrease or eliminate such problems.

VI. Genetic Transformation of Soybeans

Genetic transformation may be used to insert a selected transgene intothe soybean cultivar of the invention or may, alternatively, be used forthe preparation of transgenes which can be introduced into the cultivarof the invention by backcrossing. Methods for the transformation of manyeconomically important plants, including soybeans, are well know tothose of skill in the art. Techniques which may be employed for thegenetic transformation of soybeans include, but are not limited to,electroporation, microprojectile bombardment, Agrobacterium-mediatedtransformation and direct DNA uptake by protoplasts.

To effect transformation by electroporation, one may employ eitherfriable tissues, such as a suspension culture of cells or embryogeniccallus or alternatively one may transform immature embryos or otherorganized tissue directly. In this technique, one would partiallydegrade the cell walls of the chosen cells by exposing them topectin-degrading enzymes (pectolyases) or mechanically wound tissues ina controlled manner.

Protoplasts may also be employed for electroporation transformation ofplants (Bates, 1994; Lazzeri, 1995). For example, the generation oftransgenic soybean plants by electroporation of cotyledon-derivedprotoplasts was described by Dhir and Widholm in Intl. Patent Appl.Publ. No. WO 92/17598, the disclosure of which is specificallyincorporated herein by reference.

A particularly efficient method for delivering transforming DNA segmentsto plant cells is microprojectile bombardment. In this method, particlesare coated with nucleic acids and delivered into cells by a propellingforce. Exemplary particles include those comprised of tungsten,platinum, and preferably, gold. For the bombardment, cells in suspensionare concentrated on filters or solid culture medium. Alternatively,immature embryos or other target cells may be arranged on solid culturemedium. The cells to be bombarded are positioned at an appropriatedistance below the macroprojectile stopping plate.

An illustrative embodiment of a method for delivering DNA into plantcells by acceleration is the Biolistics Particle Delivery System, whichcan be used to propel particles coated with DNA or cells through ascreen, such as a stainless steel or Nytex screen, onto a surfacecovered with target soybean cells. The screen disperses the particles sothat they are not delivered to the recipient cells in large aggregates.It is believed that a screen intervening between the projectileapparatus and the cells to be bombarded reduces the size of projectilesaggregate and may contribute to a higher frequency of transformation byreducing the damage inflicted on the recipient cells by projectiles thatare too large.

Microprojectile bombardment techniques are widely applicable, and may beused to transform virtually any plant species. The application ofmicroprojectile bombardment for the transformation of soybeans isdescribed, for example, in U.S. Pat. No. 5,322,783, the disclosure ofwhich is specifically-incorporated herein by reference in its entirety.

Agrobacterium-mediated transfer is another widely applicable system forintroducing gene loci into plant cells. An advantage of the technique isthat DNA can be introduced into whole plant tissues, thereby bypassingthe need for regeneration of an intact plant from a protoplast. ModemAgrobacterium transformation vectors are capable of replication in E.coli as well as Agrobacterium, allowing for convenient manipulations(Klee et al., 1985). Moreover, recent technological advances in vectorsfor Agrobacterium-mediated gene transfer have improved the arrangementof genes and restriction sites in the vectors to facilitate theconstruction of vectors capable of expressing various polypeptide codinggenes. The vectors described have convenient multi-linker regionsflanked by a promoter and a polyadenylation site for direct expressionof inserted polypeptide coding genes. Additionally, Agrobacteriumcontaining both armed and disarmed Ti genes can be used fortransformation.

In those plant strains where Agrobacterium-mediated transformation isefficient, it is the method of choice because of the facile and definednature of the gene locus transfer. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art (Fraley et al., 1985; U.S. Pat. No. 5,563,055). Use ofAgrobacterium in the context of soybean transformation has beendescribed, for example, by Chee and Slightom (1995) and in U.S. Pat. No.5,569,834, the disclosures of which are specifically incorporated hereinby reference in their entirety.

Transformation of plant protoplasts also can be achieved using methodsbased on calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see, e.g.,Potrykus et al., 1985; Omirulleh et al., 1993; Fromm et al., 1986;Uchimiya et al., 1986; Marcotte et al., 1988). The demonstrated abilityto regenerate soybean plants from protoplasts makes each of thesetechniques applicable to soybean (Dhir et al., 1991).

VII. Definitions

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

A: When used in conjunction with the word "comprising" or other openlanguage in the claims, the words "a" and "an" denote "one or more."

Allele: Any of one or more alternative forms of a gene locus, all ofwhich alleles relate to one trait or characteristic. In a diploid cellor organism, the two alleles of a given gene occupy corresponding locion a pair of homologous chromosomes.

Backcrossing: A process in which a breeder repeatedly crosses hybridprogeny, for example a first generation hybrid (F₁), back to one of theparents of the hybrid progeny. Backcrossing can be used to introduce oneor more single locus conversions from one genetic background intoanother.

Brown Stem Rot: This is a visual disease score from 1 to 9 comparing allgenotypes in a given test. The score is based on leaf symptoms ofyellowing and necrosis caused by brown stem rot. A score of 1 indicatesno symptoms. Visual scores range to a score of 9 which indicates severesymptoms of leaf yellowing and necrosis.

Chromatography: A technique wherein a mixture of dissolved substancesare bound to a solid support followed by passing a column of fluidacross the solid support and varying the composition of the fluid. Thecomponents of the mixture are separated by selective elution.

Cross-pollination: Fertilization by the union of two gametes fromdifferent plants.

Crossing: The mating of two parent plants.

Diploid: A cell or organism having two sets of chromosomes.

Emasculate: The removal of plant male sex organs or the inactivation ofthe organs with a cytoplasmic or nuclear genetic factor conferring malesterility or a chemical agent.

Emergence: This is a score indicating the ability of a seed to emergefrom the soil after planting. Each genotype is given a 1 to 9 scorebased on its percent of emergence. A score of 1 indicates an excellentrate and percent of emergence, an intermediate score of 5 indicatesaverage ratings and a 9 score indicates a very poor rate and percent ofemergence.

Enzymes: Molecules which can act as catalysts in biological reactions.

F₁ Hybrid: The first generation progeny of the cross of two nonisogenicplants.

Genotype: The genetic constitution of a cell or organism.

Haploid: A cell or organism having one set of the two sets ofchromosomes in a diploid.

Iron-Deficiency Chlorosis: A plant scoring system ranging from 1 to 9based on visual observations. A score of 1 means no stunting of theplants or yellowing of the leaves and a score of 9 indicates the plantsare dead or dying caused by iron-deficiency chlorosis, a score of 5means plants have intermediate health with some leaf yellowing.

Linkage: A phenomenon wherein alleles on the same chromosome tend tosegregate together more often than expected by chance if theirtransmission was independent.

Lodging Resistance: Lodging is rated on a scale of 1 to 9. A score of 1indicates erect plants. A score of 5 indicates plants are leaning at a45 degree(s) angle in relation to the ground and a score of 9 indicatesplants are laying on the ground.

Marker: A readily detectable phenotype, preferably inherited incodominant fashion (both alleles at a locus in a diploid heterozygoteare readily detectable), with no environmental variance component, i.e.,heritability of 1.

Maturity Date: Plants are considered mature when 95% of the pods havereached their mature color. The maturity date is typically described inmeasured days from January first, which may be referred to as "JulianDays."

Phenotype: The detectable characteristics of a cell or organism, whichcharacteristics are the manifestation of gene expression.

Phytophthora Tolerance: Tolerance to Phytophthora root rot is rated on ascale of 1 to 9, with a score of 1 being the best or highest toleranceranging down to a score of 9, which indicates the plants have notolerance to Phytophthora.

Plant Height: Plant height is taken from the top of soil to the top nodeof the plant and is measured in inches.

Regeneration: The development of a plant from tissue culture.

Seed Yield (Bushels/Acre): The yield in bushels/acre is the actual yieldof the grain at harvest.

Self-pollination: The transfer of pollen from the anther to the stigmaof the same plant.

Shattering: The amount of pod dehiscence prior to harvest. Poddehiscence involves seeds falling from the pods to the soil. This is avisual score from 1 to 9 comparing all genotypes within a given test. Ascore of 1 means pods have not opened and no seeds have fallen out. Ascore of 5 indicates approximately 50% of the pods have opened, withseeds falling to the ground and a score of 9 indicates 100% of the podsare opened.

Single Locus Converted (Conversion) Plant: Plants which are developed bya plant breeding technique called backcrossing, wherein essentially allof the desired morphological and physiological characteristics of asoybean cultivar are recovered in addition to the characteristics of thesingle locus transferred into the cultivar via the backcrossingtechnique.

Tissue Culture: A composition comprising isolated cells of the same or adifferent type or a collection of such cells organized into parts of aplant.

Transgene: A genetic locus comprising a sequence which has beenintroduced into the genome of a soybean plant by transformation.

VIII. Deposit Information

A deposit of the DEKALB Genetics Corporation propriety soybean cultivarCX300, disclosed above and recited in the appended claims, has been madewith the American Type Culture Collection (ATCC), 10801 UniversityBlvd., Manassas, Va. 20110-2209. The date of deposit was Sep. 23, 1999.All restrictions upon the deposit have been removed, and the deposit isintended to meet all of the requirements of 37 C.F.R. §1.801-1.809. Theaccession number for those deposited seeds of soybean cultivar CX300 isPTA-784. The deposit will be maintained in the depository for a periodof 30 years, or 5 years after the last request, or for the effectivelife of the patent, whichever is longer, and will be replaced ifnecessary during that period.

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What is claimed is:
 1. Soybean seed designated CX300, wherein a sampleof said seed has been deposited under accession number PTA-784.
 2. Aplant produced by growing the seed of claim
 1. 3. Pollen of the plant ofclaim
 2. 4. An ovule of the plant of claim
 2. 5. A cell of the soybeanplant of claim
 2. 6. A soybean plant having all of the physiological andmorphological characteristics of the plant of claim
 2. 7. A tissueculture of regenerable cells of the soybean cultivar CX300, wherein asample of the seed of said soybean cultivar CX300 has been depositedunder accession number PTA-784.
 8. The tissue culture of claim 7,wherein the regenerable cells are embryos, meristematic cells, pollen,leaves, roots, root tips, flowers or protoplasts or callus derivedtherefrom.
 9. A soybean plant regenerated from the tissue culture ofclaim 7, wherein the regenerated soybean plant is capable of expressingall the physiological and morphological characteristics of the soybeancultivar CX300, and wherein a sample of the seed of said soybeancultivar CX300 has been deposited under accession number PTA-784. 10.The soybean plant of claim 2, further comprising a single locusconversion, wherein said soybean plant is otherwise capable ofexpressing all the physiological and morphological characteristics ofthe soybean cultivar CX300, and wherein a sample of the seed of saidsoybean cultivar CX300 has been deposited under accession numberPTA-784.
 11. The soybean plant of claim 10, wherein the single locusconversion comprises a dominant allele.
 12. The soybean plant of claim10, wherein the single locus conversion comprises a recessive allele.13. The soybean plant of claim 10, wherein the single locus was stablyinserted into a soybean genome by transformation.
 14. The soybean plantof claim 13, wherein said single locus comprises a single gene.
 15. Afirst generation (F1) hybrid soybean seed produced by crossing the plantof claim 2 with a second, distinct soybean plant.
 16. A first generationF₁ hybrid soybean plant produced by growing the seed of claim
 15. 17. Amethod of producing soybean seed, comprising crossing the soybeancultivar CX300 with itself or a second soybean plant, wherein a sampleof the seed of said soybean cultivar CX300 has been deposited underaccession number PTA-784.
 18. The method of claim 17, further defined asa method of preparing hybrid soybean seed, comprising crossing thesoybean cultivar CX300 to a second, distinct soybean plant, wherein asample of the seed of said soybean cultivar CX300 has been depositedunder accession number PTA-784.
 19. The method of claim 18, whereincrossing comprises the steps of:a) planting seed of soybean cultivarCX300 and a second, distinct soybean plant, wherein a sample of the seedof said soybean cultivar CX300 has been deposited under accession numberPTA-784; b) growing soybean plants from said seed until said plants bearflowers; c) cross pollinating a flower of said soybean cultivar CX300with pollen from said second soybean plant or cross pollinating a flowerof said second soybean plant with pollen from said soybean cultivarCX300; and d) harvesting seed resulting from said cross pollinating.